Overall Program-Abstract Blood coagulation derives from a series of specific proteolytic activation reactions that are catalyzed with narrow and defined specificity by trypsin-like serine proteinases. In several instances, these proteinases function in membrane assembled enzyme complexes. Distinctive protein substrate specificities and the modulation of enzymic function by interactions with membranes and cofactors are hallmarks of the proteolytic reactions of blood coagulation. There are major gaps in the current understanding of the molecular bases for these unique features that underlie the function of the hemostatic reactions. This program proposes an integrated approach focused on the modulation of enzymic function and specificity that uniquely arises from macromolecular interactions that underlie the action of the hemostatic enzymes. Project 1 (Krishnaswamy) uses the prothrombinase complex as a paradigm to investigate structural and functional mechanisms deriving from the membrane-dependent interactions between cofactor, proteinase and substrate in enzyme function. Project 2 (Camire) will investigate molecular mechanisms at play in the conversion of factor V to the cofactor, factor Va and the surprising new biological insights that these mechanisms reveal. Project 3 (Sullenger) employs RNA aptamers as unique probes for the macromolecular interactions essential for coagulation enzyme function and to show the way forward for novel approaches to either interfere with or enhance these interactions for therapeutic gain. Project 4 (Arruda) investigates the biochemistry of intrinsic Xase with a focus on characterizing proteinase and cofactor variants with superior function and potential for use as the next generation of therapeutics in the treatment of hemophilia. The objectives of the four projects will be supported by an administrative core (Core A), a core that provides support for molecular biology, protein expression and structural biology (Core B) and a core that supports the program with mouse models of hemostasis and thrombosis (Core C). Overall, this project applies the expertise of the individual investigators towards addressing major unanswered questions in hemostasis and thrombosis extending from biochemical insights to biological function and physiology. The proposed approaches will provide new insights into the chemistry and biology of the blood coagulation reactions with implications for an understanding of normal hemostasis and thrombosis.